Extracellular adhesive proteins

ABSTRACT

The invention provides human extracellular adhesive proteins (EXADH) and polynucleotides which identify and encode EXADH. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating or preventing disorders associated with expression of EXADH.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences ofextracellular adhesive proteins and to the use of these sequences in thediagnosis, treatment, and prevention of cancer, immune disorders, anddevelopmental disorders.

BACKGROUND OF THE INVENTION

The surface of a cell is rich in transmembrane proteoglycans,glycoproteins, glycolipids, and receptors. These macromolecules mediateadhesion with other cells and with components of the extracellularmatrix (ECM). The ECM is comprised of diverse glycoproteins,polysaccharides, and carbohydrate-binding proteins which are secretedfrom the cell and assembled into an organized meshwork in closeassociation with the cell surface. The interaction of the cell with thesurrounding matrix profoundly influences cell shape, strength,flexibility, motility, and adhesion. These dynamic properties areintimately associated with signal transduction pathways controlling cellproliferation and differentiation, tissue construction, and embryonicdevelopment.

Lectins comprise a ubiquitous family of extracellular glycoproteinswhich bind cell surface carbohydrates specifically and reversibly,resulting in the agglutination of cells. (Reviewed in Drickamer, K. andTaylor, M. E. (1993) Annu. Rev. Cell Biol. 9:237-264.) This function isparticularly important for activation of the immune response. Lectinsmediate the agglutination and mitogenic stimulation of lymphocytes atsites of inflammation. (Lasky, L. A. (1991) J. Cell. Biochem.45:139-146; Paietta, E. et al. (1989) J. Immunol. 143:2850-2857.)

Lectins are further classified into subfamilies based oncarbohydrate-binding specificity. The galectin subfamily, in particular,includes lectins that bind β-galactoside carbohydrate moieties in athioldependent manner. (Reviewed in Hadari, Y. R. et al. (1998) J. Biol.Chem. 270:3447-3453.) Galectins are widely expressed and developmentallyregulated. Because all galectins lack an N-terminal signal peptide, itis suggested that galectins are externalized through an atypicalsecretory mechanism. Two classes of galectins have been defined based onmolecular weight and oligomerization properties. Small galectins formhomodimers and are about 14 to 16 kilodaltons in mass, while largegalectins are monomeric and about 29-37 kilodaltons.

Galectins contain a characteristic carbohydrate recognition domain(CRD). The CRD is about 140 amino acids and contains several stretchesof about 1-10 amino acids which are highly conserved among allgalectins. A particular 6-amino acid motif within the CRD containsconserved tryptophan and arginine residues which are critical forcarbohydrate binding. The CRD of some galectins also contains cysteineresidues which may be important for disulfide bond formation. Secondarystructure predictions indicate that the CRD forms several β-sheets.

Galectins play a number of roles in diseases and conditions associatedwith cell-cell and cell-matrix interactions. For example, certaingalectins associate with sites of inflammation and bind to cell surfaceimmunoglobulin E molecules. In addition, galectins may play an importantrole in cancer metastasis. Galectin overexpression is correlated withthe metastatic potential of cancers in humans and mice. Moreover,anti-galectin antibodies inhibit processes associated with celltransformation, such as cell aggregation and anchorage-independentgrowth.

Prostate carcinoma tumor antigen 1 (PCTA-1) is a novel galectinimplicated in cancer progression. (Su, Z.-Z. et al. (1996) Proc. Natl.Acad. Sci. U.S.A. 93:7252-7257.) PCTA-1 was initially identified as thecell surface antigen recognized by a prostate tumor-directed monoclonalantibody, Pro 1.5. PCTA-1 cDNA is 3.85 kilobases and encodes a 317-aminoacid protein of about 35 kilodaltons. PCTA-1 is expressed in invasiveprostate carcinomas and early-stage prostate cancers, but not in normalprostate or benign prostatic hypertrophic tissue. In addition, PCTA-1 isshed from the surface of cultured prostate cancer cells into the growthmedia. Together, these results suggest that detection of PCTA-1 may beuseful for early diagnosis of prostate cancer and that levels of PCTA-1in the circulation may correlate with disease progression. In addition,preliminary studies in mice suggest that the monoclonal antibody Pro 1.5may itself be an effective therapeutic agent against tumor progression.

Fibronectin is another component of the ECM which binds to specificintegrin-family transmembrane receptors. (Reviewed in Alberts, B. et al.(1994) Molecular Biology of the Cell, Garland Publishing, New York,N.Y., pp. 986-987.) Fibronectin is found in insoluble form in connectivetissue and at the base of epithelia. Fibronectin is a dimer composed oftwo rod-shaped subunits joined by disulfide bonds. Each subunit is amultidomain glycoprotein of nearly 2500 amino acids, and each domainconsists of smaller repeated modules. The main type of module is calledthe type III fibronectin repeat, which consists of about 90-100 aminoacids which form several β-sheets. The type III repeat is found in atleast 45 protein families, most of which are involved in cell adhesion.Some type III repeats contain a specific tripeptide sequence, the RGDmotif. This motif mediates the attachment of fibronectin to the integrinreceptor. This motif is also found in various other proteins importantfor cell-cell adhesion and cell-matrix adhesion, including collagen,fibrinogen, and vitronectin.

Fibronectin is involved in a number of important functions includingwound healing, blood coagulation, cell adhesion, cell differentiation,cell migration, embryonic development, and tumor metastasis. Inaddition, the expression of fibronectin is markedly reduced inneoplastically transformed cells. Furthermore, inactivation of bothcopies of the fibronectin gene causes early embryonic lethality in mice.These knockout mice have multiple morphological defects, includingmalformation of the notochord, somites, heart, blood vessels, neuraltube, and extraembryonic structures.

A novel class of proteins involved in cell adhesion are the leucine-richrepeat proteins. Leucine rich repeats (LLRs) are about 22-28 amino acidsin length and are found in various extracellular adhesive glycoproteins.(Rothberg, J. M. et al. (1990) Genes Dev. 4:2169-2187.) LLRs arepredicted to form hydrophobic surfaces capable of interacting withmembranes. In addition, synthetic LRRs form β-sheet structures andextended filaments. (Gay, N. J. et al. (1991) FEBS Lett. 291:87-91.)These properties are consistent with a role for LRRs in cell adhesionand cell surface interactions.

The discovery of new extracellular adhesive proteins and thepolynucleotides encoding them satisfies a need in the art by providingnew compositions which are useful in the diagnosis, prevention, andtreatment of cancer, immune disorders, and developmental disorders.

SUMMARY OF THE INVENTION

The invention features substantially purified polypeptides,extracellular adhesive proteins, referred to collectively as “EXADH” andindividually as “EXADH-1” and “EXADH-2.” In one aspect, the inventionprovides a substantially purified polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,and fragments thereof.

The invention further provides a substantially purified variant havingat least 90% amino acid identity to at least one of the amino acidsequences selected from the group consisting of SEQ ID NO:1, SEQ IDNO:2, and fragments thereof. The invention also provides an isolated andpurified polynucleotide encoding the polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:2, and fragments thereof. The invention also includes an isolated andpurified polynucleotide variant having at least 70% polynucleotidesequence identity to the polynucleotide encoding the polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, and fragments thereof.

Additionally, the invention provides an isolated and purifiedpolynucleotide which hybridizes under stringent conditions to thepolynucleotide encoding the polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,and fragments thereof. The invention also provides an isolated andpurified polynucleotide having a sequence which is complementary to thepolynucleotide encoding the polypeptide comprising the amino acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,and fragments thereof.

The invention also provides an isolated and purified polynucleotidecomprising a polynucleotide sequence selected from the group consistingof SEQ ID NO:3, SEQ ID NO:4, and fragments thereof. The inventionfurther provides an isolated and purified polynucleotide variant havingat least 70% polynucleotide sequence identity to the polynucleotidesequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4,and fragments thereof. The invention also provides an isolated andpurified polynucleotide having a sequence which is complementary to thepolynucleotide comprising a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:4, and fragments thereof.

The invention also provides a method for detecting a polynucleotide in asample containing nucleic acids, the method comprising the steps of (a)hybridizing the complement of the polynucleotide sequence to at leastone of the polynucleotides of the sample, thereby forming ahybridization complex; and (b) detecting the hybridization complex,wherein the presence of the hybridization complex correlates with thepresence of a polynucleotide in the sample. In one aspect, the methodfurther comprises amplifying the polynucleotide prior to hybridization.

The invention further provides an expression vector containing at leasta fragment of the polynucleotide encoding the polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:2, and fragments thereof. In another aspect, the expressionvector is contained within a host cell.

The invention also provides a method for producing a polypeptide, themethod comprising the steps of: (a) culturing the host cell containingan expression vector containing at least a fragment of a polynucleotideunder conditions suitable for the expression of the polypeptide; and (b)recovering the polypeptide from the host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified polypeptide having the amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, andfragments thereof, in conjunction with a suitable pharmaceuticalcarrier.

The invention further includes a purified antibody which binds to apolypeptide selected from the group consisting of SEQ ID NO:1, SEQ IDNO:2, and fragments thereof. The invention also provides a purifiedagonist and a purified antagonist to the polypeptide.

The invention also provides a method for treating or preventing adisorder associated with decreased expression or activity of EXADH, themethod comprising administering to a subject in need of such treatmentan effective amount of a pharmaceutical composition comprising asubstantially purified polypeptide having the amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, andfragments thereof, in conjunction with a suitable pharmaceuticalcarrier.

The invention also provides a method for treating or preventing adisorder associated with increased expression or activity of EXADH, themethod comprising administering to a subject in need of such treatmentan effective amount of an antagonist of a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:2, and fragments thereof.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

FIGS. 1A and 1B show the amino acid sequence alignment between EXADH-1(2635136; SEQ ID NO:1) and PCTA-1 (GI 1932712; SEQ ID NO:5), producedusing the multisequence alignment program of LASERGENE software(DNASTAR, Madison Wis.).

FIG. 2 shows electronic northern analysis of SEQ ID NO:4 using theLIFESEQ sequence database (Incyte Pharmaceuticals, Palo Alto Calif.).

Table 1 shows the programs, their descriptions, references, andthreshold parameters used to analyze EXADH.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular machines, materials and methods described, as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “ahost cell” includes a plurality of such host cells, and a reference to“an antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any machines,materials, and methods similar or equivalent to those described hereincan be used to practice or test the present invention, the preferredmachines, materials and methods are now described. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, protocols, reagents and vectors which are reported inthe publications and which might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

Definitions

“EXADH” refers to the amino acid sequences of substantially purifiedEXADH obtained from any species, particularly a mammalian species,including bovine, ovine, porcine, murine, equine, and preferably thehuman species, from any source, whether natural, synthetic,semi-synthetic, or recombinant.

The term “agonist” refers to a molecule which, when bound to EXADH,increases or prolongs the duration of the effect of EXADH. Agonists mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich bind to and modulate the effect of EXADH.

An “allelic variant” is an alternative form of the gene encoding EXADH.Allelic variants may result from at least one mutation in the nucleicacid sequence and may result in altered mRNAs or in polypeptides whosestructure or function may or may not be altered. Any given natural orrecombinant gene may have none, one, or many allelic forms. Commonmutational changes which give rise to allelic variants are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

“Altered” nucleic acid sequences encoding EXADH include those sequenceswith deletions, insertions, or substitutions of different nucleotides,resulting in a polynucleotide the same as EXADH or a polypeptide with atleast one functional characteristic of EXADH. Included within thisdefinition are polymorphisms which may or may not be readily detectableusing a particular oligonucleotide probe of the polynucleotide encodingEXADH, and improper or unexpected hybridization to allelic variants,with a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding EXADH. The encoded protein may also be“altered,” and may contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent EXADH. Deliberate amino acid substitutions maybe made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues, as long as the biological or immunological activity of EXADHis retained. For example, negatively charged amino acids may includeaspartic acid and glutamic acid, positively charged amino acids mayinclude lysine and arginine, and amino acids with uncharged polar headgroups having similar hydrophilicity values may include leucine,isoleucine, and valine; glycine and alanine; asparagine and glutamine;serine and threonine; and phenylalanine and tyrosine.

The terms “amino acid” or “amino acid sequence” refer to anoligopeptide, peptide, polypeptide, or protein sequence, or a fragmentof any of these, and to naturally occurring or synthetic molecules. Inthis context, “fragments,” “immunogenic fragments,” or “antigenicfragments” refer to fragments of EXADH which are preferably at least 5to about 15 amino acids in length, most preferably at least 14 aminoacids, and which retain some biological activity or immunologicalactivity of EXADH. Where “amino acid sequence” is recited to refer to anamino acid sequence of a naturally occurring protein molecule, “aminoacid sequence” and like terms are not meant to limit the amino acidsequence to the complete native amino acid sequence associated with therecited protein molecule.

“Amplification” relates to the production of additional copies of anucleic acid sequence. Amplification is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art.

The term “antagonist” refers to a molecule which, when bound to EXADH,decreases the amount or the duration of the effect of the biological orimmunological activity of EXADH. Antagonists may include proteins,nucleic acids, carbohydrates, antibodies, or any other molecules whichdecrease the effect of EXADH.

The term “antibody” refers to intact molecules as well as to fragmentsthereof, such as Fab, F(ab′)₂, and Fv fragments, which are capable ofbinding the epitopic determinant. Antibodies that bind EXADHpolypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term “antigenic determinant” refers to that fragment of a molecule(i.e., an epitope) that makes contact with a particular antibody. When aprotein or a fragment of a protein is used to immunize a host animal,numerous regions of the protein may induce the production of antibodieswhich bind specifically to antigenic determinants (given regions orthree-dimensional structures on the protein). An antigenic determinantmay compete with the intact antigen (i.e., the immunogen used to elicitthe immune response) for binding to an antibody.

The term “antisense” refers to any composition containing a nucleic acidsequence which is complementary to the “sense” strand of a specificnucleic acid sequence. Antisense molecules may be produced by any methodincluding synthesis or transcription. Once introduced into a cell, thecomplementary nucleotides combine with natural sequences produced by thecell to form duplexes and to block either transcription or translation.The designation “negative” can refer to the antisense strand, and thedesignation “positive” can refer to the sense strand.

The term “biologically active,” refers to a protein having structural,regulatory, or biochemical functions of a naturally occurring molecule.Likewise, “immunologically active” refers to the capability of thenatural, recombinant, or synthetic EXADH, or of any oligopeptidethereof, to induce a specific immune response in appropriate animals orcells and to bind with specific antibodies.

The terms “complementary” or “complementarity” refer to the naturalbinding of polynucleotides by base pairing. For example, the sequence“5′A-G-T 3′” bonds to the complementary sequence “3′T-C-A 5′.”Complementarity between two single-stranded molecules may be “partial,”such that only some of the nucleic acids bind, or it may be “complete,”such that total complementarity exists between the single strandedmolecules. The degree of complementarity between nucleic acid strandshas significant effects on the efficiency and strength of thehybridization between the nucleic acid strands. This is of particularimportance in amplification reactions, which depend upon binding betweennucleic acids strands, and in the design and use of peptide nucleic acid(PNA) molecules.

A “composition comprising a given polynucleotide sequence” or a“composition comprising a given amino acid sequence” refer broadly toany composition containing the given polynucleotide or amino acidsequence. The composition may comprise a dry formulation or an aqueoussolution. Compositions comprising polynucleotide sequences encodingEXADH or fragments of EXADH may be employed as hybridization probes. Theprobes may be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., sodium dodecyl sulfate; SDS), and other components(e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).

“Consensus sequence” refers to a nucleic acid sequence which has beenresequenced to resolve uncalled bases, extended using XL-PCR kit(Perkin-Elmer, Norwalk Conn.) in the 5′ and/or the 3′ direction, andresequenced, or which has been assembled from the overlapping sequencesof more than one Incyte Clone using a computer program for fragmentassembly, such as the GELVIEW Fragment Assembly system (GCG, MadisonWis.). Some sequences have been both extended and assembled to producethe consensus sequence.

The term “correlates with expression of a polynucleotide” indicates thatthe detection of the presence of nucleic acids, the same or related to anucleic acid sequence encoding EXADH, by northern analysis is indicativeof the presence of nucleic acids encoding EXADH in a sample, and therebycorrelates with expression of the transcript from the polynucleotideencoding EXADH.

A “deletion” refers to a change in the amino acid or nucleotide sequencethat results in the absence of one or more amino acid residues ornucleotides.

The term “derivative” refers to the chemical modification of apolypeptide sequence, or a polynucleotide sequence. Chemicalmodifications of a polynucleotide sequence can include, for example,replacement of hydrogen by an alkyl, acyl, or amino group. A derivativepolynucleotide encodes a polypeptide which retains at least onebiological or immunological function of the natural molecule. Aderivative polypeptide is one modified by glycosylation, pegylation, orany similar process that retains at least one biological orimmunological function of the polypeptide from which it was derived.

The term “similarity” refers to a degree of complementarity. There maybe partial similarity or complete similarity. The word “identity” maysubstitute for the word “similarity.” A partially complementary sequencethat at least partially inhibits an identical sequence from hybridizingto a target nucleic acid is referred to as “substantially similar.” Theinhibition of hybridization of the completely complementary sequence tothe target sequence may be examined using a hybridization assay(Southern or northern blot, solution hybridization, and the like) underconditions of reduced stringency. A substantially similar sequence orhybridization probe will compete for and inhibit the binding of acompletely similar (identical) sequence to the target sequence underconditions of reduced stringency. This is not to say that conditions ofreduced stringency are such that non-specific binding is permitted, asreduced stringency conditions require that the binding of two sequencesto one another be a specific (i.e., a selective) interaction. Theabsence of non-specific binding may be tested by the use of a secondtarget sequence which lacks even a partial degree of complementarity(e.g., less than about 30% similarity or identity). In the absence ofnon-specific binding, the substantially similar sequence or probe willnot hybridize to the second non-complementary target sequence.

The phrases “percent identity” or “% identity” refer to the percentageof sequence similarity found in a comparison of two or more amino acidor nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MEGALIGN program (DNASTAR). TheMEGALIGN program can create alignments between two or more sequencesaccording to different methods, e.g., the clustal method. (See, e.g.,Higgins, D. G. and P. M. Sharp (1988) Gene 73:237-244.) The clustalalgorithm groups sequences into clusters by examining the distancesbetween all pairs. The clusters are aligned pairwise and then in groups.The percentage similarity between two amino acid sequences, e.g.,sequence A and sequence B, is calculated by dividing the length ofsequence A, minus the number of gap residues in sequence A, minus thenumber of gap residues in sequence B, into the sum of the residuematches between sequence A and sequence B, times one hundred. Gaps oflow or of no similarity between the two amino acid sequences are notincluded in determining percentage similarity. Percent identity betweennucleic acid sequences can also be counted or calculated by othermethods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein,J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences canalso be determined by other methods known in the art, e.g., by varyinghybridization conditions.

“Human artificial chromosomes” (HACs) are linear microchromosomes whichmay contain DNA sequences of about 6 kb to 10 Mb in size, and whichcontain all of the elements required for stable mitotic chromosomesegregation and maintenance.

The term “humanized antibody” refers to antibody molecules in which theamino acid sequence in the non-antigen binding regions has been alteredso that the antibody more closely resembles a human antibody, and stillretains its original binding ability.

“Hybridization” refers to any process by which a strand of nucleic acidbinds with a complementary strand through base pairing.

The term “hybridization complex” refers to a complex formed between twonucleic acid sequences by virtue of the formation of hydrogen bondsbetween complementary bases. A hybridization complex may be formed insolution (e.g., C₀t or R₀t analysis) or formed between one nucleic acidsequence present in solution and another nucleic acid sequenceimmobilized on a solid support (e.g., paper, membranes, filters, chips,pins or glass slides, or any other appropriate substrate to which cellsor their nucleic acids have been fixed).

The words “insertion” or “addition” refer to changes in an amino acid ornucleotide sequence resulting in the addition of one or more amino acidresidues or nucleotides, respectively, to the sequence found in thenaturally occurring molecule.

“Immune response” can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

The term “microarray” refers to an arrangement of distinctpolynucleotides on a substrate.

The terms “element” or “array element” in a microarray context, refer tohybridizable polynucleotides arranged on the surface of a substrate.

The term “modulate” refers to a change in the activity of EXADH. Forexample, modulation may cause an increase or a decrease in proteinactivity, binding characteristics, or any other biological, functional,or immunological properties of EXADH.

The phrases “nucleic acid” or “nucleic acid sequence” refer to anucleotide, oligonucleotide, polynucleotide, or any fragment thereof.These phrases also refer to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent thesense or the antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material. In this context, “fragments” refers tothose nucleic acid sequences which, when translated, would producepolypeptides retaining some functional characteristic, e.g.,antigenicity, or structural domain characteristic, e.g., ATP-bindingsite, of the full-length polypeptide.

The terms “operably associated” or “operably linked” refer tofunctionally related nucleic acid sequences. A promoter is operablyassociated or operably linked with a coding sequence if the promotercontrols the translation of the encoded polypeptide. While operablyassociated or operably linked nucleic acid sequences can be contiguousand in the same reading frame, certain genetic elements, e.g., repressorgenes, are not contiguously linked to the sequence encoding thepolypeptide but still bind to operator sequences that control expressionof the polypeptide.

The term “oligonucleotide” refers to a nucleic acid sequence of at leastabout 6 nucleotides to 60 nucleotides, preferably about 15 to 30nucleotides, and most preferably about 20 to 25 nucleotides, which canbe used in PCR amplification or in a hybridization assay or microarray.“Oligonucleotide” is substantially equivalent to the terms “amplimer,”“primer,” “oligomer,” and “probe,” as these terms are commonly definedin the art.

“Peptide nucleic acid” (PNA) refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide of at least about 5nucleotides in length linked to a peptide backbone of amino acidresidues ending in lysine. The terminal lysine confers solubility to thecomposition. PNAs preferentially bind complementary single stranded DNAor RNA and stop transcript elongation, and may be pegylated to extendtheir lifespan in the cell.

The term “sample” is used in its broadest sense. A sample suspected ofcontaining nucleic acids encoding EXADH, or fragments thereof, or EXADHitself, may comprise a bodily fluid; an extract from a cell, chromosome,organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA,or cDNA, in solution or bound to a substrate; a tissue; a tissue print;etc.

The terms “specific binding” or “specifically binding” refer to thatinteraction between a protein or peptide and an agonist, an antibody, oran antagonist. The interaction is dependent upon the presence of aparticular structure of the protein, e.g., the antigenic determinant orepitope, recognized by the binding molecule. For example, if an antibodyis specific for epitope “A,” the presence of a polypeptide containingthe epitope A, or the presence of free unlabeled A, in a reactioncontaining free labeled A and the antibody will reduce the amount oflabeled A that binds to the antibody.

The term “stringent conditions” refers to conditions which permithybridization between polynucleotides and the claimed polynucleotides.Stringent conditions can be defined by salt concentration, theconcentration of organic solvent, e.g., formamide, temperature, andother conditions well known in the art. In particular, stringency can beincreased by reducing the concentration of salt, increasing theconcentration of formamide, or raising the hybridization temperature.

The term “substantially purified” refers to nucleic acid or amino acidsequences that are removed from their natural environment and areisolated or separated, and are at least about 60% free, preferably about75% free, and most preferably about 90% free from other components withwhich they are naturally associated.

A “substitution” refers to the replacement of one or more amino acids ornucleotides by different amino acids or nucleotides, respectively.

“Substrate” refers to any suitable rigid or semi-rigid support includingmembranes, filters, chips, slides, wafers, fibers, magnetic ornonmagnetic beads, gels, tubing, plates, polymers, microparticles andcapillaries. The substrate can have a variety of surface forms, such aswells, trenches, pins, channels and pores, to which polynucleotides orpolypeptides are bound.

“Transformation” describes a process by which exogenous DNA enters andchanges a recipient cell. Transformation may occur under natural orartificial conditions according to various methods well known in theart, and may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod for transformation is selected based on the type of host cellbeing transformed and may include, but is not limited to, viralinfection, electroporation, heat shock, lipofection, and particlebombardment. The term “transformed” cells includes stably transformedcells in which the inserted DNA is capable of replication either as anautonomously replicating plasmid or as part of the host chromosome, aswell as transiently transformed cells which express the inserted DNA orRNA for limited periods of time.

A “variant” of EXADH polypeptides refers to an amino acid sequence thatis altered by one or more amino acid residues. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties (e.g., replacement of leucine withisoleucine). More rarely, a variant may have “nonconservative” changes(e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, LASERGENE software (DNASTAR).

The term “variant,” when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to EXADH. Thisdefinition may also include, for example, “allelic” (as defined above),“splice,” “species,” or “polymorphic” variants. A splice variant mayhave significant identity to a reference molecule, but will generallyhave a greater or lesser number of polynucleotides due to alternatesplicing of exons during mRNA processing. The corresponding polypeptidemay possess additional functional domains or an absence of domains.Species variants are polynucleotide sequences that vary from one speciesto another. The resulting polypeptides generally will have significantamino acid identity relative to each other. A polymorphic variant is avariation in the polynucleotide sequence of a particular gene betweenindividuals of a given species. Polymorphic variants also may encompass“single nucleotide polymorphisms” (SNPs) in which the polynucleotidesequence varies by one base. The presence of SNPs may be indicative of,for example, a certain population, a disease state, or a propensity fora disease state.

The Invention

The invention is based on the discovery of new human extracellularadhesive proteins (EXADH), the polynucleotides encoding EXADH, and theuse of these compositions for the diagnosis, treatment, or prevention ofcancer, immune disorders, and developmental disorders.

Nucleic acids encoding the EXADH-1 of the present invention were firstidentified in Incyte Clone 2635136 from the tibial periosteum cDNAlibrary (BONTNOT01) using a computer search, e.g., BLAST, for amino acidsequence alignments. A consensus sequence, SEQ ID NO:3, was derived fromthe following overlapping and/or extended nucleic acid sequences: IncyteClones 2635136H1 (BONTNOT01), 485976R6 (HNT2RAT01), 002834R6 and002834T6 (HMC1NOT01), and 2818607T6 (BRSTNOT14).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1. EXADH-1 is 336 amino acids inlength and has one potential cAMP- and cGMP-dependent protein kinasephosphorylation site at S132; four potential casein kinase IIphosphorylation sites at S24, S164, S242, and S257; and four potentialprotein kinase C phosphorylation sites at T102, S205, S242, and T253.EXADH-1 has several features characteristic of galectin proteins. Thepredicted molecular weight of EXADH-1 is about 37.5 kilodaltons,consistent with the size of large monomeric galectins. BLOCKS and PFAManalyses indicate that the region of EXADH-1 from G67 to D174 is similarto galectin CRD. In particular, the conserved tryptophan and arginineresidues which are critical for carbohydrate binding are conserved inEXADH-1 at W117 and R122. EXADH-1 contains seven cysteine residues, fourof which are in the putative CRD. Furthermore, secondary structureanalysis indicates that the N-terminus of EXADH-1 inclusive of theputative CRD is predicted to form several β-sheets. As shown in FIGS. 1Aand 1B, EXADH-1 has chemical and structural similarity with human PCTA-1(GI 1932712; SEQ ID NO:5). In particular, EXADH-1 and PCTA-1 share 28%identity overall and 44% identity within the putative CRD of EXADH-1from G67 to D174. In addition, sequence alignments among EXADH-1 andnine galectin CRD sequences indicate that EXADH-1 contains at least 46out of 55 conserved CRD residues in the region from P48 to D174. Afragment of SEQ ID NO:3 from about nucleotide 346 to about nucleotide375 is useful as a hybridization probe. Northern analysis shows theexpression of this sequence in various libraries, at least 62% of whichare associated with cancer, cell proliferation, or fetal development andat least 38% of which are associated with the immune response. Inparticular, 38% of the libraries expressing this sequence are derivedfrom reproductive tissue and 19% are derived from hematopoietic tissue.

Nucleic acids encoding the EXADH-2 of the present invention were firstidentified in Incyte Clone 2687731 from the lung cDNA library(LUNGNOT23) using a computer search, e.g., BLAST, for amino acidsequence alignments. A consensus sequence, SEQ ID NO:4, was derived fromthe following overlapping and/or extended nucleic acid sequences: IncyteClones 2687731H1 (LUNGNOT23), 268773X309F1, 268773X328F1, and268773X326F1 (HNT2NOT01), 2836031H1 (TLYMNOT03), 3035425H1 (TLYMNOT05),2929080T6 (TLYMNOT04), 3003158H1 (TLYMNOT06), and 2107346H1 (BRAITUT03).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:2. EXADH-2 is 70 amino acids inlength and has nine potential N-glycosylation sites at N93, N103, N223,N382, N522, N579, N608, N624, and N625; five potential casein kinase 11phosphorylation sites at S51, T95, S242, T468, and T487; ten potentialprotein kinase C phosphorylation sites at T42, S173, T470, T487, S540,S544, S551, S568, T610, and S693; and one potential tyrosine kinasephosphorylation site at Y578. EXADH-2 contains a potential RGD cellattachment sequence from R277 to D279. PRINTS analysis indicates thatleucine-rich repeat signatures are interspersed throughout theN-terminal half of EXADH-2. PRINTS and PFAM analyses indicate that theregions of EXADH-2 from N521 to K604 and from E226 to Y244 are similarto fibronectin type III repeats. A signal peptide is predicted from M1to A22. A fragment of SEQ ID NO:4 from about nucleotide 129 to aboutnucleotide 158 is useful as a hybridization probe. Northern analysisshows the expression of this sequence in various libraries, at least 65%of which are associated with cancer, cell proliferation, or fetaldevelopment and at least 35% of which are associated with the immuneresponse. In particular, 41 % of the libraries expressing this sequenceare derived from neural tissue and 29% are derived from hematopoietictissue. FIG. 2 shows the four cDNA libraries in which SEQ ID NO:4 ismost abundantly expressed. Abundance refers to the number of times SEQID NO:4 appears in each of the libraries listed, and percent abundancerefers to the abundance divided by the total number of sequencesexamined in a given library. Of particular note is that the percentabundance of SEQ ID NO:4 is highest in cDNA libraries derived fromlymphocytes. Sixteen other cDNA libraries express SEQ ID NO:4 at levelswhich range from about 3- to 25-fold lower.

The invention also encompasses EXADH variants. A preferred EXADH variantis one which has at least about 80%, more preferably at least about 90%,and most preferably at least about 95% amino acid sequence identity tothe EXADH amino acid sequence, and which contains at least onefunctional or structural characteristic of EXADH.

The invention also encompasses polynucleotides which encode EXADH. In aparticular embodiment, the invention encompasses a polynucleotidesequence comprising a sequence selected from the group consisting of SEQID NO:3 and SEQ ID NO:4, which encodes EXADH.

The invention also encompasses a variant of a polynucleotide sequenceencoding EXADH. In particular, such a variant polynucleotide sequencewill have at least about 70%, more preferably at least about 85%, andmost preferably at least about 95% polynucleotide sequence identity tothe polynucleotide sequence encoding EXADH. A particular aspect of theinvention encompasses a variant of a polynucleotide sequence comprisinga sequence selected from the group consisting of SEQ ID NO:3 and SEQ IDNO:4 which has at least about 70%, more preferably at least about 85%,and most preferably at least about 95% polynucleotide sequence identityto a nucleic acid sequence selected from the group consisting of SEQ IDNO:3 and SEQ ID NO:4. Any one of the polynucleotide variants describedabove can encode an amino acid sequence which contains at least onefunctional or structural characteristic of EXADH.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding EXADH, some bearing minimal similarity to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring EXADH, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode EXADH and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring EXADH under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding EXADH or its derivatives possessing a substantially differentcodon usage, e.g., inclusion of non-naturally occurring codons. Codonsmay be selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding EXADH and its derivatives without altering the encoded aminoacid sequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

The invention also encompasses production of DNA sequences which encodeEXADH and EXADH derivatives, or fragments thereof, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents well known in the art. Moreover, synthetic chemistry may beused to introduce mutations into a sequence encoding EXADH or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO:3, SEQ ID NO:4 and fragmentsthereof under various conditions of stringency. (See, e.g., Wahl, G. M.and S. L. Berger (1987) Methods Enzymol. 152:3-407; Kimmel, A. R. (1987)Methods Enzymol. 152:507-511.) For example, stringent salt concentrationwill ordinarily be less than about 750 mM NaCl and 75 mM trisodiumcitrate, preferably less than about 500 mM NaCl and 50 mM trisodiumcitrate, and most preferably less than about 250 mM NaCl and 25 mMtrisodium citrate. Low stringency hybridization can be obtained in theabsence of organic solvent, e.g., formamide, while high stringencyhybridization can be obtained in the presence of at least about 35%formamide, and most preferably at least about 50% formamide. Stringenttemperature conditions will ordinarily include temperatures of at leastabout 30° C., more preferably of at least about 37° C., and mostpreferably of at least about 42° C. Varying additional parameters, suchas hybridization time, the concentration of detergent, e.g., sodiumdodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA,are well known to those skilled in the art. Various levels of stringencyare accomplished by combining these various conditions as needed. In apreferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl,75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment,hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodiumcitrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA(ssDNA). In a most preferred embodiment, hybridization will occur at 42°C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and200 μg/ml ssDNA. Useful variations on these conditions will be readilyapparent to those skilled in the art.

The washing steps which follow hybridization can also vary instringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude temperature of at least about 25° C., more preferably of atleast about 42° C., and most preferably of at least about 68° C. In apreferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and1.0% SDS. In a most preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art.

Methods for DNA sequencing are well known in the art and may be used topractice any of the embodiments of the invention. The methods may employsuch enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (U.S.Biochemical, Cleveland Ohio), Taq polymerase (Perkin-Elmer),thermostable T7 polymerase (Amersham Pharmacia Biotech, PiscatawayN.J.), or combinations of polymerases and proofreading exonucleases suchas those found in the ELONGASE amplification system (Life Technologies,Gaithersburg Md.). Preferably, sequence preparation is automated withmachines such as the Hamilton MICROLAB 2200 (Hamilton, Reno Nev.),Peltier Thermal Cycler 200 (PTC200; MJ Research, Watertown Mass.) andthe ABI CATALYST 800 (Perkin-Elmer). Sequencing is then carried outusing either ABI 373 or 377 DNA Sequencing Systems (Perkin-Elmer) or theMEGABACE capillary electrophoresis system (Molecular Dynamics, SunnyvaleCalif.). The resulting sequences are analyzed using a variety ofalgorithms which are well known in the art. (See, e.g., Ausubel, F. M.(1997) Short Protocols in Molecular Biology, John Wiley & Sons, New YorkN.Y., unit 7.7; Meyers, R. A. (1995) Molecular Biology andBiotechnology, Wiley VCH, New York N.Y., pp. 856-853.)

The nucleic acid sequences encoding EXADH may be extended utilizing apartial nucleotide sequence and employing various PCR-based methodsknown in the art to detect upstream sequences, such as promoters andregulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method,inverse PCR, uses primers that extend in divergent directions to amplifyunknown sequence from a circularized template. The template is derivedfrom restriction fragments comprising a known genomic locus andsurrounding sequences. (See, e.g., Triglia, T. et al. (1988) NucleicAcids Res. 16:8186.) A third method, capture PCR, involves PCRamplification of DNA fragments adjacent to known sequences in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to insert anengineered double-stranded sequence into a region of unknown sequencebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art. (See, e.g., Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR,nested primers, and PROMOTERFINDER libraries (Clontech, Palo AltoCalif.) to walk genomic DNA. This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO 4.06 Primer Analysis software (NationalBiosciences, Plymouth Minn.) or another appropriate program, to be about22 to 30 nucleotides in length, to have a GC content of about 50% ormore, and to anneal to the template at temperatures of about 68° C. to72° C.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. In addition,random-primed libraries, which often include sequences containing the 5′regions of genes, are preferable for situations in which an oligo d(T)library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5′ non-transcribed regulatoryregions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentnucleotide-specific, laser-stimulated fluorescent dyes, and a chargecoupled device camera for detection of the emitted wavelengths.Output/light intensity may be converted to electrical signal usingappropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR,Perkin-Elmer), and the entire process from loading of samples tocomputer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable forsequencing small DNA fragments which may be present in limited amountsin a particular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode EXADH may be cloned in recombinant DNAmolecules that direct expression of EXADH, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express EXADH.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alterEXADH-encoding sequences for a variety of purposes including, but notlimited to, modification of the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example,oligonucleotide-mediated site-directed mutagenesis may be used tointroduce mutations that create new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, and so forth.

In another embodiment, sequences encoding EXADH may be synthesized, inwhole or in part, using chemical methods well known in the art. (See,e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, and Hom, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232.)Alternatively, EXADH itself or a fragment thereof may be synthesizedusing chemical methods. For example, peptide synthesis can be performedusing various solid-phase techniques. (See, e.g., Roberge, J. Y. et al.(1995) Science 269:202-204.) Automated synthesis may be achieved usingthe ABI 431 A Peptide Synthesizer (Perkin-Elmer). Additionally, theamino acid sequence of EXADH, or any part thereof, may be altered duringdirect synthesis and/or combined with sequences from other proteins, orany part thereof, to produce a variant polypeptide.

The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g, Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182:392-421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, T. (1984) Proteins. Structures andMolecular Properties, WH Freeman, New York N.Y.)

In order to express a biologically active EXADH, the nucleotidesequences encoding EXADH or derivatives thereof may be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for transcriptional and translational control of theinserted coding sequence in a suitable host. These elements includeregulatory sequences, such as enhancers, constitutive and induciblepromoters, and 5′ and 3′ untranslated regions in the vector and inpolynucleotide sequences encoding EXADH. Such elements may vary in theirstrength and specificity. Specific initiation signals may also be usedto achieve more efficient translation of sequences encoding EXADH. Suchsignals include the ATG initiation codon and adjacent sequences, e.g.the Kozak sequence. In cases where sequences encoding EXADH and itsinitiation codon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used. (See,e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding EXADH andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning. A Laboratory Manual, Cold Spring HarborPress, Plainview N.Y., ch. 4, 8, and 16-17; Ausubel, F. M. et al. (1995)Current Protocols in Molecular Biology, John Wiley & Sons, New YorkN.Y., ch. 9, 13, and 16.)

A variety of expression vector/host systems may be utilized to containand express sequences encoding EXADH. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

In bacterial systems, a number of cloning and expression vectors may beselected depending upon the use intended for polynucleotide sequencesencoding EXADH. For example, routine cloning, subcloning, andpropagation of polynucleotide sequences encoding EXADH can be achievedusing a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene,La Jolla Calif.) or pSPORT1 plasmid (Life Technologies). Ligation ofsequences encoding EXADH into the vector's multiple cloning sitedisrupts the lacZ gene, allowing a colorimetric screening procedure foridentification of transformed bacteria containing recombinant molecules.In addition, these vectors may be useful for in vitro transcription,dideoxy sequencing, single strand rescue with helper phage, and creationof nested deletions in the cloned sequence. (See, e.g., Van Heeke, G.and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When largequantities of EXADH are needed, e.g. for the production of antibodies,vectors which direct high level expression of EXADH may be used. Forexample, vectors containing the strong, inducible T5 or T7 bacteriophagepromoter may be used.

Yeast expression systems may be used for production of EXADH. A numberof vectors containing constitutive or inducible promoters, such as alphafactor, alcohol oxidase, and PGH, may be used in the yeast Saccharomycescerevisiae or Pichia pastoris. In addition, such vectors direct eitherthe secretion or intracellular retention of expressed proteins andenable integration of foreign sequences into the host genome for stablepropagation. (See, e.g., Ausubel, 1995, supra; Grant et al. (1987)Methods Enzymol. 153:516-54; and Scorer, C. A. et al. (1994)Bio/Technology 12:181-184.)

Plant systems may also be used for expression of EXADH. Transcription ofsequences encoding EXADH may be driven viral promoters, e.g., the 35Sand 19S promoters of CaMV used alone or in combination with the omegaleader sequence from TMV. (Takamatsu, N. (1987) EMBO J. 6:307-311.)Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be is used. (See, e.g., Coruzzi, G. et al.(1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ.17:85-105.) These constructs can be introduced into plant cells bydirect DNA transformation or pathogen-mediated transfection. (See, e.g.,The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill,New York N.Y., pp. 191-196.)

In mammalian cells, a number of viral-based expression systems may beutilized. In cases where an adenovirus is used as an expression vector,sequences encoding EXADH may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses EXADH in host cells. (See, e.g., Logan, J. and T. Shenk (1984)Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells. SV40 or EBV-based vectorsmay also be used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained in and expressed from aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes. (See, e.g., Harrington, J. J. etal. (1997) Nat Genet. 15:345-355.) For long term production ofrecombinant proteins in mammalian systems, stable expression of EXADH incell lines is preferred. For example, sequences encoding EXADH can betransfonned into cell lines using expression vectors which may containviral origins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for about 1to 2 days in enriched media before being switched to selective media.The purpose of the selectable marker is to confer resistance to aselective agent, and its presence allows growth and recovery of cellswhich successfully express the introduced sequences. Resistant clones ofstably transformed cells may be propagated using tissue culturetechniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase and adenine phosphoribosyltransferase genes, for use intk⁻ or apr⁻ cells, respectively. (See, e.g., Wigler, M. et al. (1977)Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate; neo confers resistance to the aminoglycosides neomycin andG-418; and als or pat confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M.et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F.et al. (1981) J. Mol. Biol. 150:1-14) Additional selectable genes havebeen described, e.g., trpB and hisD, which alter cellular requirementsfor metabolites. (See, e.g., Hartman, S. C. and R. C. Mulligan (1988)Proc. Natl. Acad. Sci. 85:8047-8051.) Visible markers, e.g.,anthocyanins, green fluorescent proteins (GFP; Clontech), βglucuronidase and its substrate β-glucuronide, or luciferase and itssubstrate luciferin may be used. These markers can be used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system.(See, e.g., Rhodes, C. A. (1995) Methods Mol. Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingEXADH is inserted within a marker gene sequence, transformed cellscontaining sequences encoding EXADH can be identified by the absence ofmarker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding EXADH under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the tandem gene as well.

In general, host cells that contain the nucleic acid sequence encodingEXADH and that express EXADH may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCRamplification, and protein bioassay or immunoassay techniques whichinclude membrane, solution, or chip based technologies for the detectionand/or quantification of nucleic acid or protein sequences.

Immunological methods for detecting and measuring the expression ofEXADH using either specific polyclonal or monoclonal antibodies areknown in the art. Examples of such techniques include enzyme-linkedimmunosorbent assays (ELISAs), radioimmunoassays (RIAs), andfluorescence activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on EXADH is preferred, but a competitivebinding assay may be employed. These and other assays are well known inthe art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, aLaboratory Manual, APS Press, St Paul Minn., Sect. IV; Coligan, J. E. etal. (1997) Current Protocols in Immunology, Greene Pub. Associates andWiley-Interscience, New York NY; and Pound, J. D. (1998) ImmunochemicalProtocols, Humana Press, Totowa N.J.).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding EXADH includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encoding EXADH,or any fragments thereof, may be cloned into a vector for the productionof an mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits, such as those provided by AmershamPharmacia Biotech, Promega (Madison Wis.), and U.S. Biochemical.Suitable reporter molecules or labels which may be used for ease ofdetection include radionuclides, enzymes, fluorescent, chemiluminescent,or chromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding EXADH may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or retained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeEXADH may be designed to contain signal sequences which direct secretionof EXADH through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to specify protein targeting, folding, and/oractivity. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and W138), are available from the American TypeCulture Collection (ATCC, Bethesda Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding EXADH may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric EXADHprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of EXADH activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the EXADH encodingsequence and the heterologous protein sequence, so that EXADH may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel (1995, supra, ch 10). A variety of commercially available kitsmay also be used to facilitate expression and purification of fusionproteins.

In a further embodiment of the invention, synthesis of radiolabeledEXADH may be achieved in vitro using the TNT rabbit reticulocyte lysateor wheat germ extract systems (Promega). These systems coupletranscription and translation of protein-coding sequences operablyassociated with the T7, T3, or SP6 promoters. Translation takes place inthe presence of a radiolabeled amino acid precursor, preferably³⁵S-methionine.

Fragments of EXADH may be produced not only by recombinant production,but also by direct peptide synthesis using solid-phase techniques. (See,e.g., Creighton, supra pp. 55-60.) Protein synthesis may be performed bymanual techniques or by automation. Automated synthesis may be achieved,for example, using the ABI 431 A Peptide Synthesizer (Perkin-Elmer).Various fragments of EXADH may be synthesized separately and thencombined to produce the full length molecule.

Therapeutics

Chemical and structural similarity exists among regions of EXADH-1,PCTA-1, and sequences conserved among galectins. Chemical and structuralsimilarity also exists among regions of EXADH-2, fibronectin type IIImodules, and LRRs. In addition, EXADH is expressed in cells and celllines associated with fetal tissue, cancer, and the immune system.Therefore, EXADH appears to be associated with cancer, immune disorders,and developmental disorders. In the treatment of cancer, immunedisorders, and developmental disorders associated with increased EXADHactivity, it is desirable to decrease the expression or activity ofEXADH. In the treatment of the above conditions associated withdecreased EXADH activity, it is desirable to provide the protein or toincrease the expression of EXADH. Therefore, in one embodiment, EXADH ora fragment or derivative thereof may be administered to a subject totreat or prevent a disorder associated with decreased expression oractivity of EXADH. Such disorders can include, but are not limited to, acancer such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,sarcoma, teratocarcinoma, and, in particular, cancers of the adrenalgland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; an immune disorder such asacquired immunodeficiency syndrome (AIDS), Addison's disease, adultrespiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, bronchitis, cholecystitis, contactdermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections, trauma, X-linkedagammaglobinemia of Bruton, common variable immunodeficiency (CVI),DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgAdeficiency, severe combined immunodeficiency disease (SCID),immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrichsyndrome), Chediak-Higashi syndrome, chronic granulomatous diseases,hereditary angioneurotic edema, and immunodeficiency associated withCushing's disease; and a developmental disorder such as renal tubularacidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenneand Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGRsyndrome (Wilms' tumor, aniridia, genitourinary abnormalities, andmental retardation), Smith-Magenis syndrome, myelodysplastic syndrome,hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditaryneuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,hypothyroidism, hydrocephalus, seizure disorders such as Syndenham'schorea and cerebral palsy, spina bifida, anencephaly,craniorachischisis, congenital glaucoma, cataract, sensorineural hearingloss, and any disorder associated with cell growth and differentiation,embryogenesis, and morphogenesis involving any tissue, organ, or systemof a subject, e.g., the brain, adrenal gland, kidney, skeletal orreproductive system.

In another embodiment, a vector capable of expressing EXADH or afragment or derivative thereof may be administered to a subject to treator prevent a disorder associated with decreased expression or activityof EXADH including, but not limited to, those described above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified EXADH in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a disorder associated with decreased expression or activity ofEXADH including, but not limited to, those provided above.

In still another embodiment, an agonist which modulates the activity ofEXADH may be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of EXADH including, butnot limited to, those listed above.

In a further embodiment, an antagonist of EXADH may be administered to asubject to treat or prevent a disorder associated with increasedexpression or activity of EXADH. Such disorders may include, but are notlimited to, those discussed above. In one aspect, an antibody whichspecifically binds EXADH may be used directly as an antagonist orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express EXADH.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding EXADH may be administered to a subject to treator prevent a disorder associated with increased expression or activityof EXADH including, but not limited to, those described above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of EXADH may be produced using methods which are generallyknown in the art. In particular, purified EXADH may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind EXADH. Antibodies to EXADH may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are especially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith EXADH or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to EXADH have an amino acid sequence consisting of atleast about 5 amino acids, and, more preferably, of at least about 10amino acids. It is also preferable that these oligopeptides, peptides,or fragments are identical to a portion of the amino acid sequence ofthe natural protein and contain the entire amino acid sequence of asmall, naturally occurring molecule. Short stretches of EXADH aminoacids may be fused with those of another protein, such as KLH, andantibodies to the chimeric molecule may be produced.

Monoclonal antibodies to EXADH may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce EXADH-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci.88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for EXADH mayalso be generated. For example, such fragments include, but are notlimited to, F(ab′)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 246:1275-1281.)

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between EXADH and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering EXADH epitopes is preferred, but a competitivebinding assay may also be employed (Pound, supra).

In another embodiment of the invention, the polynucleotides encodingEXADH, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding EXADH may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding EXADH. Thus, complementary molecules orfragments may be used to modulate EXADH activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments can bedesigned from various locations along the coding or control regions ofsequences encoding EXADH.

Expression vectors derived from retroviruses, adenoviruses, or herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue, or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors to express nucleic acid sequencescomplementary to the polynucleotides encoding EXADH. (See, e.g.,Sambrook, supra; Ausubel, 1995, supra.)

Genes encoding EXADH can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide,or fragment thereof, encoding EXADH. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5′, or regulatory regions of the gene encodingEXADH. Oligonucleotides derived from the transcription initiation site,e.g., between about positions −10 and +10 from the start site, arepreferred. Similarly, inhibition can be achieved using triple helixbase-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee, J. E. et al. (1994)in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,Futura Publishing, Mt. Kisco N.Y., pp. 163-177.) A complementarysequence or antisense molecule may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingEXADH.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding EXADH. SuchDNA sequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA, constitutivelyor inducibly, can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the molecule,or the use of phosphorothioate or 2′O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art. (See, e.g., Goldman, C. K. et al. (1997) NatureBiotechnology 15:462466.)

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical or sterile composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of EXADH,antibodies to EXADH, and mimetics, agonists, antagonists, or inhibitorsof EXADH. The compositions may be administered alone or in combinationwith at least one other agent, such as a stabilizing compound, which maybe administered in any sterile, biocompatible pharmaceutical carrierincluding, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs, or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing, Easton Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with fillers or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, or synthetic fatty acid esters, such asethyl oleate, triglycerides, or liposomes. Non-lipid polycationic aminopolymers may also be used for delivery. Optionally, the suspension mayalso contain suitable stabilizers or agents to increase the solubilityof the compounds and allow for the preparation of highly concentratedsolutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tendto be more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of EXADH, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells or inanimal models such as mice, rats, rabbits, dogs, or pigs. An animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example EXADH or fragments thereof, antibodies of EXADH,and agonists, antagonists or inhibitors of EXADH, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD50 (the doselethal to 50% of the population) statistics. The dose ratio oftherapeutic to toxic effects is the therapeutic index, and it can beexpressed as the ED₅₀/LD₅₀ ratio. Pharmaceutical compositions whichexhibit large therapeutic indices are preferred. The data obtained fromcell culture assays and animal studies are used to formulate a range ofdosage for human use. The dosage contained in such compositions ispreferably within a range of circulating concentrations that includesthe ED₅₀ with little or no toxicity. The dosage varies within this rangedepending upon the dosage form employed, the sensitivity of the patient,and the route of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Longacting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind EXADH may beused for the diagnosis of disorders characterized by expression ofEXADH, or in assays to monitor patients being treated with EXADH oragonists, antagonists, or inhibitors of EXADH. Antibodies useful fordiagnostic purposes may be prepared in the same manner as describedabove for therapeutics. Diagnostic assays for EXADH include methodswhich utilize the antibody and a label to detect EXADH in human bodyfluids or in extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by covalent ornon-covalent attachment of a reporter molecule. A wide variety ofreporter molecules, several of which are described above, are known inthe art and may be used.

A variety of protocols for measuring EXADH, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of EXADH expression. Normal or standard values for FXADHexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toEXADH under conditions suitable for complex formation. The amount ofstandard complex formation may be quantitated by various methods,preferably by photometric means. Quantities of EXADH expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingEXADH may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofEXADH may be correlated with disease. The diagnostic assay may be usedto determine absence, presence, and excess expression of EXADH, and tomonitor regulation of EXADH levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding EXADH or closely related molecules may be used to identifynucleic acid sequences which encode EXADH. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding EXADH, allelicvariants, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably have at least 50% sequence identity to any of theEXADH encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequence of SEQID NO:3 or SEQ ID NO:4 or from genomic sequences including promoters,enhancers, and introns of the EXADH gene.

Means for producing specific hybridization probes for DNAs encodingEXADH include the cloning of polynucleotide sequences encoding EXADH orEXADH derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, are commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotide sequences encoding EXADH may be used for the diagnosis ofdisorders associated with expression of EXADH. Examples of suchdisorders include, but are not limited to, a cancer such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; an immune disorder such asacquired immunodeficiency syndrome (AIDS), Addison's disease, adultrespiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, bronchitis, cholecystitis, contactdermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderima, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections, trauma, X-linkedagammaglobinemia of Bruton, common variable immunodeficiency (CVI),DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgAdeficiency, severe combined immunodeficiency disease (SCID),immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrichsyndrome), Chediak-Higashi syndrome, chronic granulomatous diseases,hereditary angioneurotic edema, and immunodeficiency associated withCushing's disease; and a developmental disorder such as renal tubularacidosis, anemia, Cushing's syndrome, achondroplastic dwarfism, Duchenneand Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGRsyndrome (Wilms' tumor, aniridia, genitourinary abnormalities, andmental retardation), Smith-Magenis syndrome, myelodysplastic syndrome,hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditaryneuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,hypothyroidism, hydrocephalus, seizure disorders such as Syndenham'schorea and cerebral palsy, spina bifida, anencephaly,craniorachischisis, congenital glaucoma, cataract, sensorineural hearingloss, and any disorder associated with cell growth and differentiation,embryogenesis, and morphogenesis involving any tissue, organ, or systemof a subject, e.g., the brain, adrenal gland, kidney, skeletal orreproductive system. The polynucleotide sequences encoding EXADH may beused in Southern or northern analysis, dot blot, or other membrane-basedtechnologies; in PCR technologies; in dipstick, pin, and ELISA assays;and in microarrays utilizing fluids or tissues from patients to detectaltered EXADH expression. Such qualitative or quantitative methods arewell known in the art.

In a particular aspect, the nucleotide sequences encoding EXADH may beuseful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingEXADH may be labeled by standard methods and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the patient sample is significantlyaltered in comparison to a control sample then the presence of alteredlevels of nucleotide sequences encoding EXADH in the sample indicatesthe presence of the associated disorder. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or to monitor the treatment of anindividual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of EXADH, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding EXADH, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding EXADH may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding EXADH, or a fragment of a polynucleotide complementary to thepolynucleotide encoding EXADH, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of EXADHinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves.(See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244;Duplaa, C. et al. (1993) Anal. Biochem. 229-236.) The speed ofquantitation of multiple samples may be accelerated by running the assayin an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or colorimetric responsegives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used astargets in a microarray. The microarray can be used to monitor theexpression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

Microarrays may be prepared, used, and analyzed using methods known inthe art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci.93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)

In another embodiment of the invention, nucleic acid sequences encodingEXADH may be used to generate hybridization probes useful in mapping thenaturally occurring genomic sequence. The sequences may be mapped to aparticular chromosome, to a specific region of a chromosome, or toartificial chromosome constructions, e.g., human artificial chromosomes(HACs), yeast artificial chromosomes (YACs), bacterial artificialchromosomes (BACs), bacterial P1 constructions, or single chromosomecDNA libraries. (See, e.g., Harrington, J. J. et al. (1997) Nat Genet.15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134; and Trask, B. J.(1991) Trends Genet. 7:149-154.)

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical chromosome mapping techniques and genetic map data. (See, e.g.,Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.) Examples ofgenetic map data can be found in various scientific journals or at theOnline Mendelian Inheritance in Man (OMIM) site. Correlation between thelocation of the gene encoding EXADH on a physical chromosomal map and aspecific disorder, or a predisposition to a specific disorder, may helpdefine the region of DNA associated with that disorder. The nucleotidesequences of the invention may be used to detect differences in genesequences among normal, carrier, and affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms by physical mapping. This provides valuable informationto investigators searching for disease genes using positional cloning orother gene discovery techniques. Once the disease or syndrome has beencrudely localized by genetic linkage to a particular genomic region,e.g., ataxia-telangiectasia to 11 q22-23, any sequences mapping to thatarea may represent associated or regulatory genes for furtherinvestigation. (See, e.g., Gatti, R. A. et al. (1988) Nature336:577-580.) The nucleotide sequence of the subject invention may alsobe used to detect differences in the chromosomal location due totranslocation, inversion, etc., among normal, carrier, or affectedindividuals.

In another embodiment of the invention, EXADH, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, bome on a cell surface, or locatedintracellularly. The formation of binding complexes between EXADH andthe agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate. The test compounds arereacted with EXADH, or fragments thereof, and washed. Bound EXADH isthen detected by methods well known in the art. Purified EXADH can alsobe coated directly onto plates for use in the aforementioned drugscreening techniques. Alternatively, non-neutralizing antibodies can beused to capture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding EXADH specificallycompete with a test compound for binding EXADH. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with EXADH.

In additional embodiments, the nucleotide sequences which encode EXADHmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I. Construction of cDNA Libraries

The BONTNOT01 cDNA library was constructed using RNA isolated fromtibial periosteum removed from a 20-year-old Caucasian male during ahemipelvectomy with amputation above the knee. Pathology for theassociated tumor tissue indicated partially necrotic and cysticosteoblastic grade 3 osteosarcoma, post-chemotherapy. Family historyincluded osteogenesis imperfecta, closed fracture, and type II diabetes.

The LUNGNOT23 cDNA library was constructed using RNA isolated from leftlobe lung tissue removed from a 58-year-old Caucasian male. Pathologyfor the associated tumor tissue indicated metastatic grade 3 (of 4)osteosarcoma. Patient history included soft tissue cancer, secondarycancer of the lung, prostate cancer, and an acute duodenal ulcer withhemorrhage. Family history included prostate cancer, breast cancer, andacute leukemia.

For construction of the BONTNOT01 and LUNGNOT23 cDNA libraries, frozentissue from each of the above sources was homogenized and lysed inTRIzol reagent (1 gm tissue/10 ml TRIzol; Life Technologies), amonophasic solution of phenol and guanidine isothiocyanate, using aBrinkmann Homogenizer Polytron PT-3000 (Brinkmann Instruments, WestburyN.Y.). After brief incubation on ice, chloroform was added (1:5 v/v),and the mixture was centrifuged to separate the phases. The upperaqueous phase was removed to a fresh tube, and isopropanol was added toprecipitate RNA. The RNA was resuspended in RNase-free water and treatedwith DNase. The RNA was re-extracted as necessary with acidphenol-chloroform to increase purity, and the RNA was reprecipitatedwith sodium acetate and ethanol.

From each RNA preparation, poly(A+) RNA was isolated using the OLIGOTEXkit (QIAGEN, Chatsworth Calif.). Poly(A+) RNA was used for cDNAsynthesis and construction of each cDNA library according to therecommended protocols in the SUPERSCRIPT plasmid system (LifeTechnologies). The cDNAs were fractionated on a SEPHAROSE CL4B column(Amersham Pharmacia Biotech), and those cDNAs exceeding 400 bp wereligated into pINCY (Incyte Pharmaceuticals). Recombinant plasmids weretransformed into DH5α competent cells (Life Technologies).

II. Isolation of cDNA Clones

Plasmid DNA was released from the cells and purified using the REAL Prep96 plasmid kit (QIAGEN). The recommended protocol was employed exceptfor the following changes: 1) the bacteria were cultured in 1 ml ofsterile Terrific Broth (Life Technologies) with carbenicillin at 25 mg/land glycerol at 0.4%; 2) after the cultures were incubated for 19 hours,the cells were lysed with 0.3 ml of lysis buffer; and 3) followingisopropanol precipitation, the plasmid DNA pellets were each resuspendedin 0.1 ml of distilled water. The DNA samples were stored at 4° C.

III. Sequencing and Analysis

The cDNAs were prepared for sequencing using either an ABI CATALYSTS 800(Perkin-Elmer) or a MICROLAB 2200 (Hamilton) sequencing preparationsystem in combination with PTC-200 thermal cyclers (MJ Research). ThecDNAs were sequenced using the ABI PRISM 373 or 377 sequencing systemsand ABI protocols, base calling software, and kits (Perkin-Elmer).Alternatively, solutions and dyes from Amersham Pharmacia Biotech wereused. Reading frames were determined using standard methods (Ausubel,1995, supra). Some of the cDNA sequences were selected for extensionusing the techniques disclosed in Example V.

The polynucleotide sequences derived from cDNA, extension, and shotgunsequencing were assembled and analyzed using a combination of softwareprograms which utilize algorithms well known to those skilled in theart. Table 1 summarizes the software programs, descriptions, references,and threshold parameters used. The first column of Table 1 shows thetools, programs, and algorithms used, the second column provides a briefdescription thereof, the third column presents the references which areincorporated by reference herein, and the fourth column presents, whereapplicable, the scores, probability values, and other parameters used toevaluate the strength of a match between two sequences (the higher theprobability the greater the homology). Sequences were analyzed usingMACDNASIS PRO software (Hitachi Software Engineering, S. San FranciscoCalif.) and LASERGENE software (DNASTAR).

The polynucleotide sequences were validated by removing vector, linker,and polyA sequences and by masking ambiguous bases, using algorithms andprograms based on BLAST, dynamic programing, and dinucleotide nearestneighbor analysis. The sequences were then queried against a selectionof public databases such as GenBank primate, rodent, mammalian,vertebrate, and eukaryote databases, and BLOCKS to acquire annotation,using programs based on BLAST, FASTA, and BLIMPS. The sequences wereassembled into full length polynucleotide sequences using programs basedon Phred, Phrap, and Consed, and were screened for open reading framesusing programs based on GeneMark, BLAST, and FASTA. The full lengthpolynucleotide sequences were translated to derive the correspondingfull length amino acid sequences, and these full length sequences weresubsequently analyzed by querying against databases such as the GenBankdatabases (described above), SwissProt, BLOCKS, PRINTS, PFAM, andProsite.

IV. Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7;Ausubel, 1995, supra, ch. 4 and 16.)

Analogous computer techniques applying BLAST were used to search foridentical or related molecules in nucleotide databases such as GenBankor LIFESEQ database (Incyte Pharmaceuticals). This analysis is muchfaster than multiple membrane-based hybridizations. In addition, thesensitivity of the computer search can be modified to determine whetherany particular match is categorized as exact or similar. The basis ofthe search is the product score, which is defined as:

sequence identity×% maximum BLAST score/100

The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. For example,with a product score of 40, the match will be exact within a 1% to 2%error, and, with a product score of 70, the match will be exact. Similarmolecules are usually identified by selecting those which show productscores between 15 and 40, although lower scores may identify relatedmolecules.

The results of northern analyses are reported as a list of libraries inwhich the transcript encoding EXADH occurred. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a CDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the CDNA library. Further analyses produced the percentagevalues of tissue-specific and disease expression which are reported inthe description of the invention.

V. Extension of EXADH Encoding Polynucleotides

The nucleic acid sequences of Incyte ESTs 2633156 and 2687731 were usedto design oligonucleotide primers for extending partial nucleotidesequences to full length. For each nucleic acid sequence, one primer wassynthesized to initiate extension of an antisense polynucleotide, andthe other was synthesized to initiate extension of a sensepolynucleotide. Primers were used to facilitate the extension of theknown sequence “outward” which generates amplicons containing newunknown nucleotide sequence for the region of interest. The initialprimers were designed from the cDNA using OLIGO 4.06 software (NationalBiosciences), or another appropriate program, to be about 22 to 30nucleotides in length, to have a GC content of about 50% or more, and toanneal to the target sequence at temperatures of about 68° C. to about72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations was avoided.

Selected human cDNA libraries (Life Technologies) were used to extendthe sequence. If more than one extension is necessary or desired,additional sets of primers are designed to further extend the knownregion.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR kit (PerkinElmer) and thoroughly mixing the enzyme andreaction mix. PCR was performed using the PTC200 thermal cycler (M.J.Research) beginning with 40 pmol of each primer and the recommendedconcentrations of all other components of the kit, with the followingparameters:

Step 1 94° C. for 1 min (initial denaturation) Step 2 65° C. for 1 minStep 3 68° C. for 6 min Step 4 94° C. for 15 sec Step 5 65° C. for 1 minStep 6 68° C. for 7 min Step 7 Repeat steps 4-6 for an additional 15cycles Step 8 94° C. for 15 sec Step 9 65° C. for 1 min Step 10 68° C.for 7:15 min Step 11 Repeat steps 8-10 for an additional 12 cycles Step12 72° C. for 8 min Step 13 4° C. (and holding)

A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6% to 0.8%) agarosemini-gel to determine which reactions were successful in extending thesequence. Bands thought to contain the largest products were excisedfrom the gel, purified using the QIAQUICK kit (QIAGEN), and trimmed ofoverhangs using Klenow enzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2 to 3 hours, or overnight at 16° C. Competent E. colicells (in 40 μl of appropriate media) were transformed with 3 μl ofligation mixture and cultured in 80 μl of SOC medium. (See, e.g.,Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37°C., the E. coli mixture was plated on Luria Bertani (LB) agar (See,e.g., Sambrook, supra, Appendix A, p. 1) containing carbenicillin (2×carb). The following day, several colonies were randomly picked fromeach plate and cultured in 150 μl of liquid LB/2× carb medium placed inan individual well of an appropriate commercially-available sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and, afterdilution 1:10 with water, 5 μl from each sample was transferred into aPCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55° C. for 30sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for an additional29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (and holding)

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequences of SEQ ID NO:3 and SEQ ID NO:4are used to obtain 5′ regulatory sequences using the procedure above,oligonucleotides designed for 5′ extension, and an appropriate genomiclibrary.

VI. Labeling and use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:3 and SEQ ID NO:4 areemployed to screen cDNAs, genomic DNAs, or mRNAs. Although the labelingof oligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [γ-³²P] adenosinetriphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase(DuPont NEN, Boston Mass.). The labeled oligonucleotides aresubstantially purified using a SEPHADEX G-25 superfine size exclusiondextran bead column (Amersham Pharmacia Biotech). An aliquot containing10⁷ counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba1,or Pvu II (DuPont NEN).

The DNA from each digest is fractionated on a 0.7% agarose gel andtransferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT-AR film(Eastman Kodak, Rochester N.Y.) is exposed to the blots to film forseveral hours, hybridization patterns are compared visually.

VII. Microarrays

A chemical coupling procedure and an ink jet device can be used tosynthesize array elements on the surface of a substrate. (See, e.g.,Baldeschweiler, supra.) An array analogous to a dot or slot blot mayalso be used to arrange and link elements to the surface of a substrateusing thermal, UV, chemical, or mechanical bonding procedures. A typicalarray may be produced by hand or using available methods and machinesand contain any appropriate number of elements. After hybridization,nonhybridized probes are removed and a scanner used to determine thelevels and patterns of fluorescence. The degree of complementarity andthe relative abundance of each probe which hybridizes to an element onthe microarray may be assessed through analysis of the scanned images.

Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereofmay comprise the elements of the microarray. Fragments suitable forhybridization can be selected using software well known in the art suchas LASERGENE software (DNASTAR). Full-length cDNAs, ESTs, or fragmentsthereof corresponding to one of the nucleotide sequences of the presentinvention, or selected at random from a cDNA library relevant to thepresent invention, are arranged on an appropriate substrate, e.g., aglass slide. The CDNA is fixed to the slide using, e.g., UVcross-linking followed by thermal and chemical treatments and subsequentdrying. (See, e.g., Schena, M. et al. (1995) Science 270:467-470;Shalon, D. et al. (1996) Genome Res. 6:639-645.) Fluorescent probes areprepared and used for hybridization to the elements on the substrate.The substrate is analyzed by procedures described above.

VIII. Complementary Polynucleotides

Sequences complementary to the EXADH-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring EXADH. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 software(National Biosciences) and the coding sequence of EXADH. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5′ sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the EXADH-encoding transcript.

IX. Expression of EXADH

Expression and purification of EXADH is achieved using bacterial orvirus-based expression systems. For expression of EXADH in bacteria,cDNA is subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21(DE3). Antibiotic resistant bacteria express EXADH uponinduction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expressionof EXADH in eukaryotic cells is achieved by infecting insect ormammalian cell lines with recombinant Autographica californica nuclearpolyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding EXADH by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus. (See Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. U.S.A. 91:3224-3227; Sandig, V. et al.(1996) Hum. Gene Ther. 7:1937-1945.)

In most expression systems, EXADH is synthesized as a fusion proteinwith, e.g., glutathione Stransferase (GST) or a peptide epitope tag,such as FLAG or 6-His, permitting rapid, single-step, affinity-basedpurification of recombinant fusion protein from crude cell lysates. GST,a 26-kilodalton enzyme from Schistosoma japonicum, enables thepurification of fusion proteins on immobilized glutathione underconditions that maintain protein activity and antigenicity (AmershamPharmacia Biotech). Following purification, the GST moiety can beproteolytically cleaved from EXADH at specifically engineered sites.FLAG, an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak). 6-His, a stretch of six consecutive histidine residues,enables purification on metal-chelate resins (QIAGEN). Methods forprotein expression and purification are discussed in Ausubel (1995,supra, ch 10 and 16). Purified EXADH obtained by these methods can beused directly in the following activity assay.

X. Demonstration of EXADH Activity

An assay for EXADH activity measures the amount of cell aggregationinduced by overexpression of EXADH. In this assay, cultured cells suchas NIH3T3 are transfected with cDNA encoding EXADH contained within asuitable mammalian expression vector under control of a strong promoter.Cotransfection with cDNA encoding a fluorescent marker protein, such asGreen Fluorescent Protein (Clontech), is useful for identifying stabletransfectants. The amount of cell agglutination, or clumping, associatedwith transfected cells is compared with that associated withuntransfected cells. The amount of cell agglutination is a directmeasure of EXADH activity.

XI. Functional Assays

EXADH function is assessed by expressing the sequences encoding EXADH atphysiologically elevated levels in mammalian cell culture systems. cDNAis subcloned into a mammalian expression vector containing a strongpromoter that drives high levels of cDNA expression. Vectors of choiceinclude pCMV SPORT (Life Technologies) and pCR3.1 (Invitrogen, CarlsbadCalif.), both of which contain the cytomegalovirus promoter. 5-10 μg ofrecombinant vector are transiently transfected into a human cell line,preferably of endothelial or hematopoietic origin, using either liposomeformulations or electroporation. 1-2 μg of an additional plasmidcontaining sequences encoding a marker protein are cotransfected.Expression of a marker protein provides a means to distinguishtransfected cells from nontransfected cells and is a reliable predictorof cDNA expression from the recombinant vector. Marker proteins ofchoice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64,or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laseroptics-based technique, is used to identify transfected cells expressingGFP or CD64-GFP, and to evaluate properties, for example, theirapoptotic state. FCM detects and quantifies the uptake of fluorescentmolecules that diagnose events preceding or coincident with cell death.These events include changes in nuclear DNA content as measured bystaining of DNA with propidium iodide; changes in cell size andgranularity as measured by forward light scatter and 90 degree sidelight scatter; down-regulation of DNA synthesis as measured by decreasein bromodeoxyuridine uptake; alterations in expression of cell surfaceand intracellular proteins as measured by reactivity with specificantibodies; and alterations in plasma membrane composition as measuredby the binding of fluorescein-conjugated Annexin V protein to the cellsurface. Methods in flow cytometry are discussed in Ormerod, M. G.(1994) Flow Cytometry, Oxford, New York N.Y.

The influence of EXADH on gene expression can be assessed using highlypurified populations of cells transfected with sequences encoding EXADHand either CD64 or CD64-GFP. CD64 and CD64GFP are expressed on thesurface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding EXADH and other genes of interestcan be analyzed by northern analysis or microarray techniques.

XII. Production of EXADH Specific Antibodies

EXADH substantially purified using polyacrylamide gel electrophoresis(PAGE; see, e.g., Harrington, M. G. (1990) Methods Enzymol.182:488-495), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols.

Alternatively, the EXADH amino acid sequence is analyzed using LASERGENEsoftware (DNASTAR) to determine regions of high immunogenicity, and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Methods for selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions are well described in the art. (See, e.g., Ausubel,1995, supra, ch. 11.) Typically, oligopeptides 15 residues in length aresynthesized using an ABI 431 A Peptide Synthesizer (Perkin-Elmer) usingfmoc-chemistry and coupled to KLH (Sigma-Aldrich, St. Louis Mo.) byreaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) toincrease immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits areimmunized with the oligopeptide-KLH complex in complete Freund'sadjuvant. Resulting antisera are tested for antipeptide activity by, forexample, binding the peptide to plastic, blocking with 1% BSA, reactingwith rabbit antisera, washing, and reacting with radio-iodinated goatanti-rabbit IgG.

XIII. Purification of Naturally Occurring EXADH Using SpecificAntibodies

Naturally occurring or recombinant EXADH is substantially purified byimmunoaffinity chromatography using antibodies specific for EXADH. Animmunoaffinity column is constructed by covalently coupling anti-EXADHantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing EXADH are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of EXADH (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/EXADH binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andEXADH is collected.

XIV. Identification of Molecules Which Interact with EXADH

EXADH, or biologically active fragments thereof, are labeled with ¹²⁵IBolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled EXADH, washed, and anywells with labeled EXADH complex are assayed. Data obtained usingdifferent concentrations of EXADH are used to calculate values for thenumber, affinity, and association of EXADH with the candidate molecules.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

TABLE 1 Program Description Reference Parameter Threshold ABI FACTURA Aprogram that removes vector sequences and masks Perkin-Elmer AppliedBiosystems, ambiguous bases in nucleic acid sequences. Foster City, CA.ABI/PARACEL FDF A Fast Data Finder useful in comparing and annotatingPerkin-Elmer Applied Biosystems, Mismatch <50% amino acid or nucleicacid sequences. Foster City, CA; Paracel Inc., Pasadena, CA. ABIAutoAssembler A program that assembles nucleic acid sequences.Perkin-Elmer Applied Biosystems, Foster City, CA. BLAST A Basic LocalAlignment Search Tool useful in sequence Altschul, S.F. et al. (1990) J.Mol. Biol. ESTs: Probability value = 1.0E − 8 similarity search foramino acid and nucleic acid sequences. 215:403-410; Altschul, S.F. etal. (1997) or less BLAST includes five functions: blastp, blastn,blastx, Nucleic Acids Res. 25: 3389-3402. Full Length sequences:Probability tblastn, and tblastx. value = 1.0E − 10 or less FASTA APearson and Lipman algorithm that searches for similarity Pearson, W.R.and D.J. Lipman (1988) Proc. ESTs: fasta E value = 1.06E − 6 between aquery sequence and a group of sequences of the Natl. Acad Sci.85:2444-2448; Pearson, W.R. Assembled ESTs: fasta Identity = same type.FASTA comprises as least five functions: fasta, (1990) Methods Enzymol.183: 63-98; and 95% or greater and Match tfasta, fastx, tfastx, andssearch. Smith, T.F. and M.S. Waterman (1981) Adv. length = 200 bases orgreater; fastx Appl. Math. 2:482-489. E value = 1.0E − 8 or less FullLength sequences: fastx score = 100 or greater BLIMPS A BLocks IMProvedSearcher that matches a sequence Henikoff, S and J.G. Henikoff, Nucl.Acid Res., Score = 1000 or greater; Ratio of against those in BLOCKS andPRINTS databases to search 19:6565-72, 1991. J.G. Henikoff and S.Score/Strength = 0.75 or larger; for gene families, sequence homology,and structural Henikoff (1996) Methods Enzymol. 266:88-105; andProbability value = 1.0E − 3 or fingerprint regions. and Attwood, T.K.et al. (1997) J. Chem. Inf. less Comput. Sci. 37: 417-424. PFAM A HiddenMarkov Models-based application useful for Krogh, A. et al. (1994) J.Mol. Biol., 235:1501- Score = 10-50 bits, depending on protein familysearch. 1531; Sonnhammer, E.L.L. et al. (1988) individual proteinfamilies Nucleic Acids Res. 26:320-322. ProfileScan An algorithm thatsearches for structural and sequence Gribskov, M. et al. (1988) CABIOS4:61-66; Score = 4.0 or greater motifs in protein sequences that matchsequence patterns Gribskov, et al. (1989) Methods Enzymol. defined inProsite. 183:146-159; Bairoch, A. et al. (1997) Nucleic Acids Res. 25:217-221. Phred A base-calling algorithm that examines automated Ewing,B. et al. (1998) Genome sequencer traces with high sensitivity andprobability Res. 8:175-185; Ewing B. and P. Green (1998) Genome Res.8:186- 194. Phrap A Phils Revised Assembly Program including SWAT andSmith, T.F. and M.S. Waterman (1981) Adv. Score = 120 or greater; MatchCrossMatch, programs based on efficient implementation of Appl. Math.2:482-489; Smith, T.F. and M.S. length = 56 or greater theSmith-Waterman algorithm, useful in searching Waterman (1981) J. Mol.Biol. 147:195-197; sequence homology and assembling DNA sequences. andGreen, P., University of Washington, Seattle, WA. Consed A graphicaltool for viewing and editing Phrap assemblies Gordon, D. et al. (1998)Genome Res. 8:195-202. SPScan A weight matrix analysis program thatscans protein Nielson, H. et al. (1997) Protein Engineering Score = 5 orgreater sequences for the presence of secretory signal peptides. 10:1-6;Claverie, J.M. and S. Audic (1997) CABIOS 12: 431-439. Motifs A programthat searches amino acid sequences for patterns Bairoch et al. supra;Wisconsin that matched those defined in Prosite. Package Program Manual,version 9, page M51-59, Genetics Computer Group, Madison, WI.

5 1 336 PRT Homo sapiens 2635136 1 Met Ser Gln Pro Ser Gly Gly Arg AlaPro Gly Thr Arg Ile Tyr 1 5 10 15 Ser Trp Ser Cys Pro Thr Val Met SerPro Gly Glu Lys Leu Asp 20 25 30 Pro Ile Pro Asp Ser Phe Ile Leu Gln ProPro Val Phe His Pro 35 40 45 Val Val Pro Tyr Val Thr Thr Ile Phe Gly GlyLeu His Ala Gly 50 55 60 Lys Met Val Met Leu Gln Gly Val Val Pro Leu AspAla His Arg 65 70 75 Phe Gln Val Asp Phe Gln Cys Gly Cys Ser Leu Cys ProArg Pro 80 85 90 Asp Ile Ala Phe His Phe Asn Pro Arg Phe His Thr Thr LysPro 95 100 105 His Val Ile Cys Asn Thr Leu His Gly Gly Arg Trp Gln ArgGlu 110 115 120 Ala Arg Trp Pro His Leu Ala Leu Arg Arg Gly Ser Ser PheLeu 125 130 135 Ile Leu Phe Leu Phe Gly Asn Glu Glu Val Lys Val Ser ValAsn 140 145 150 Gly Gln His Phe Leu His Phe Arg Tyr Arg Leu Pro Leu SerHis 155 160 165 Val Asp Thr Leu Gly Ile Phe Gly Asp Ile Leu Val Glu AlaVal 170 175 180 Gly Phe Leu Asn Ile Asn Pro Phe Val Glu Gly Ser Arg GluTyr 185 190 195 Pro Ala Gly His Pro Phe Leu Leu Met Ser Pro Arg Leu GluVal 200 205 210 Pro Cys Ser His Ala Leu Pro Gln Gly Leu Ser Pro Gly GlnVal 215 220 225 Ile Ile Val Arg Gly Leu Val Leu Gln Glu Pro Lys His PheThr 230 235 240 Val Ser Leu Arg Asp Gln Ala Ala His Ala Pro Val Thr LeuArg 245 250 255 Ala Ser Phe Ala Asp Arg Thr Leu Ala Trp Ile Ser Arg TrpGly 260 265 270 Gln Lys Lys Leu Ile Ser Ala Pro Phe Leu Phe Tyr Pro GlnArg 275 280 285 Phe Phe Glu Val Leu Leu Leu Phe Gln Glu Gly Gly Leu LysLeu 290 295 300 Ala Leu Asn Gly Gln Gly Leu Gly Ala Thr Ser Met Asn GlnGln 305 310 315 Ala Leu Glu Gln Leu Arg Glu Leu Arg Ile Ser Gly Ser ValGln 320 325 330 Leu Tyr Cys Val His Ser 335 2 708 PRT Homo sapiens2687731 2 Met Lys Asp Met Pro Leu Arg Ile His Val Leu Leu Gly Leu Ala 15 10 15 Ile Thr Thr Leu Val Gln Ala Val Asp Lys Lys Val Asp Cys Pro 2025 30 Arg Leu Cys Thr Cys Glu Ile Arg Pro Trp Phe Thr Pro Arg Ser 35 4045 Ile Tyr Met Glu Ala Ser Thr Val Asp Cys Asn Asp Leu Gly Leu 50 55 60Leu Thr Phe Pro Ala Arg Leu Pro Ala Asn Thr Gln Ile Leu Leu 65 70 75 LeuGln Thr Asn Asn Ile Ala Lys Ile Glu Tyr Ser Thr Asp Phe 80 85 90 Pro ValAsn Leu Thr Gly Leu Asp Leu Ser Gln Asn Asn Leu Ser 95 100 105 Ser ValThr Asn Ile Asn Val Lys Lys Met Pro Gln Leu Leu Ser 110 115 120 Val TyrLeu Glu Glu Asn Lys Leu Thr Glu Leu Pro Glu Lys Cys 125 130 135 Leu SerGlu Leu Ser Asn Leu Gln Glu Leu Tyr Ile Asn His Asn 140 145 150 Leu LeuSer Thr Ile Ser Pro Gly Ala Phe Ile Gly Leu His Asn 155 160 165 Leu LeuArg Leu His Leu Asn Ser Asn Arg Leu Gln Met Ile Asn 170 175 180 Ser LysTrp Phe Asp Ala Leu Pro Asn Leu Glu Ile Leu Met Ile 185 190 195 Gly GluAsn Pro Ile Ile Arg Ile Lys Asp Met Asn Phe Lys Pro 200 205 210 Leu IleAsn Leu Arg Ser Leu Val Ile Ala Gly Ile Asn Leu Thr 215 220 225 Glu IlePro Asp Asn Ala Leu Val Gly Leu Glu Asn Leu Glu Ser 230 235 240 Ile SerPhe Tyr Asp Asn Arg Leu Ile Lys Val Pro His Val Ala 245 250 255 Leu GlnLys Val Val Asn Leu Lys Phe Leu Asp Leu Asn Lys Asn 260 265 270 Pro IleAsn Arg Ile Arg Arg Gly Asp Phe Ser Asn Met Leu His 275 280 285 Leu LysGlu Leu Gly Ile Asn Asn Met Pro Glu Leu Ile Ser Ile 290 295 300 Asp SerLeu Ala Val Asp Asn Leu Pro Asp Leu Arg Lys Ile Glu 305 310 315 Ala ThrAsn Asn Pro Arg Leu Ser Tyr Ile His Pro Asn Ala Phe 320 325 330 Phe ArgLeu Pro Lys Leu Glu Ser Leu Met Leu Asn Ser Asn Ala 335 340 345 Leu SerAla Leu Tyr His Gly Thr Ile Glu Ser Leu Pro Asn Leu 350 355 360 Lys GluIle Ser Ile His Ser Asn Pro Ile Arg Cys Asp Cys Val 365 370 375 Ile ArgTrp Met Asn Met Asn Lys Thr Asn Ile Arg Phe Met Glu 380 385 390 Pro AspSer Leu Phe Cys Val Asp Pro Pro Glu Phe Gln Gly Gln 395 400 405 Asn ValArg Gln Val His Phe Arg Asp Met Met Glu Ile Cys Leu 410 415 420 Pro LeuIle Ala Pro Glu Ser Phe Pro Ser Asn Leu Asn Val Glu 425 430 435 Ala GlySer Tyr Val Ser Phe His Cys Arg Ala Thr Ala Glu Pro 440 445 450 Gln ProGlu Ile Tyr Trp Ile Thr Pro Ser Gly Gln Lys Leu Leu 455 460 465 Pro AsnThr Leu Thr Asp Lys Phe Tyr Val His Ser Glu Gly Thr 470 475 480 Leu AspIle Asn Gly Val Thr Pro Lys Glu Gly Gly Leu Tyr Thr 485 490 495 Cys IleAla Thr Asn Leu Val Gly Ala Asp Leu Lys Ser Val Met 500 505 510 Ile LysVal Asp Gly Ser Phe Pro Gln Asp Asn Asn Gly Ser Leu 515 520 525 Asn IleLys Ile Arg Asp Ile Gln Ala Asn Ser Val Leu Val Ser 530 535 540 Trp LysAla Ser Ser Lys Ile Leu Lys Ser Ser Val Lys Trp Thr 545 550 555 Ala PheVal Lys Thr Glu Asn Ser His Ala Ala Gln Ser Ala Arg 560 565 570 Ile ProSer Asp Val Lys Val Tyr Asn Leu Thr His Leu Asn Pro 575 580 585 Ser ThrGlu Tyr Lys Ile Cys Ile Asp Ile Pro Thr Ile Tyr Gln 590 595 600 Lys AsnArg Lys Lys Cys Val Asn Val Thr Thr Lys Gly Leu His 605 610 615 Pro AspGln Lys Glu Tyr Glu Lys Asn Asn Thr Thr Thr Leu Met 620 625 630 Ala CysLeu Gly Gly Leu Leu Gly Ile Ile Gly Val Ile Cys Leu 635 640 645 Ile SerCys Leu Ser Pro Glu Met Asn Cys Asp Gly Gly His Ser 650 655 660 Tyr ValArg Asn Tyr Leu Gln Lys Pro Thr Phe Ala Leu Gly Glu 665 670 675 Leu TyrPro Pro Leu Ile Asn Leu Trp Glu Ala Gly Lys Glu Lys 680 685 690 Ser ThrSer Leu Lys Val Lys Ala Thr Val Ile Gly Leu Pro Thr 695 700 705 Asn MetSer 3 1643 DNA Homo sapiens 2635136 3 tgcaatggcc atatgctgca gacccggagtgggtagttag ttggttaatg ccagtcttcc 60 tcccctggac actgagttct gctgacagcccccgcccagc cagagctctg ctgtatacca 120 ccgggagtgg ggctggtgtg gagcctggaggtcgcccgct gccctcctag ggctgctcca 180 gacagcatta aaacgctgca ggtcgcaggtgagactaaca gctgggagag ctgctccagg 240 catttaggac cctgactggg gcagatgagtcagcccagtg ggggcagggc tcctggaacg 300 aggatctaca gttggagttg ccccactgtcatgtcacctg gagaaaaact ggacccaatt 360 cctgacagct tcattctgca accaccagtcttccacccgg tggttcctta tgtcacgacg 420 atttttggag gcctgcatgc aggcaagatggtcatgctgc aaggagtggt ccctctagat 480 gcacacaggt ttcaggtgga cttccagtgtggctgcagcc tgtgtccccg gccagatatc 540 gccttccact tcaaccctcg cttccataccaccaagcccc atgtcatctg caacaccctg 600 catggtggac gctggcaaag ggaggcccggtggccccacc tggccctgcg aagaggctcc 660 agcttcctca tcctctttct cttcgggaatgaggaagtga aggtgagtgt gaatggacag 720 cactttctcc acttccgcta ccggctcccactgtctcatg tggacacgct gggtatattt 780 ggtgacatcc tggtagaggc tgttggattcctgaacatca atccatttgt ggagggcagc 840 agagagtacc cagctggaca tcctttcctgctgatgagcc ccaggctgga ggtgccctgc 900 tcacatgctc ttccccaggg tctctcgcctgggcaggtca tcatagtacg gggactggtc 960 ttgcaagagc cgaagcattt tactgtgagcctgagggacc aggctgccca tgctcctgtg 1020 acactcaggg cctccttcgc agacagaactctggcctgga tctcccgctg ggggcagaag 1080 aaactgatct cagccccctt cctcttttacccccagagat tctttgaggt gctgctcctg 1140 ttccaggagg gagggctgaa gctggcgctcaatgggcagg ggctgggggc caccagcatg 1200 aaccagcagg ccctggagca gctgcgggagctccggatca gtggaagtgt ccagctctac 1260 tgtgtccact cctgaggatg gttccagggaaataccgcca gaaaacaaga aggtcagccc 1320 actcccaggg ccccactctc ctcccctcattaaaccatcc acctgacacc agcacatcag 1380 gcctggttca cctctggggt cacgagactgagtctacagg agctttgggc ctgagggaag 1440 gcacaagagt gcaaaggttc ctcgaactctgcaccttcct ccaccaggag cctgggatat 1500 ggctccatct gccttcaggg cctggactgcactcacagag gcaagtgttg tagactaaca 1560 aagatactcc aaaatacaat ggcttaaagaatgtggtcat ttattcttta ttatttattt 1620 atttgtggtc aaataaataa ata 1643 42290 DNA Homo sapiens 2687731 4 cttactagca ctgactgtgg aatccttaagggcccattac atttctgaag aagaaagcta 60 agatgaagga catgccactc cgaattcatgtgctacttgg cctagctatc actacactag 120 tacaagctgt agataaaaaa gtggattgtccacggttatg tacgtgtgaa atcaggcctt 180 ggtttacacc cagatccatt tatatggaagcatctacagt ggattgtaat gatttaggtc 240 ttttaacttt cccagccaga ttgccagctaacacacagat tcttctccta cagactaaca 300 atattgcaaa aattgaatac tccacagactttccagtaaa ccttactggc ctggatttat 360 ctcaaaacaa tttatcttca gtcaccaatattaatgtaaa aaagatgcct cagctccttt 420 ctgtgtacct agaggaaaac aaacttactgaactgcctga aaaatgtctg tccgaactga 480 gcaacttaca agaactctat attaatcacaacttgctttc tacaatttca cctggagcct 540 ttattggcct acataatctt cttcgacttcatctcaattc aaatagattg cagatgatca 600 acagtaagtg gtttgatgct cttccaaatctagagattct gatgattggg gaaaatccaa 660 ttatcagaat caaagacatg aactttaagcctcttatcaa tcttcgcagc ctggttatag 720 ctggtataaa cctcacagaa ataccagataacgccttggt tggactggaa aacttagaaa 780 gcatctcttt ttacgataac aggcttattaaagtacccca tgttgctctt caaaaagttg 840 taaatctcaa atttttggat ctaaataaaaatcctattaa tagaatacga aggggtgatt 900 ttagcaatat gctacactta aaagagttggggataaataa tatgcctgag ctgatttcca 960 tcgatagtct tgctgtggat aacctgccagatttaagaaa aatagaagct actaacaacc 1020 ctagattgtc ttacattcac cccaatgcatttttcagact ccccaagctg gaatcactca 1080 tgctgaacag caatgctctc agtgccctgtaccatggtac cattgagtct ctgccaaacc 1140 tcaaggaaat cagcatacac agtaaccccatcaggtgtga ctgtgtcatc cgttggatga 1200 acatgaacaa aaccaacatt cgattcatggagccagattc actgttttgc gtggacccac 1260 ctgaattcca aggtcagaat gttcggcaagtgcatttcag ggacatgatg gaaatttgtc 1320 tccctcttat agctcctgag agctttccttctaatctaaa tgtagaagct gggagctatg 1380 tttcctttca ctgtagagct actgcagaaccacagcctga aatctactgg ataacacctt 1440 ctggtcaaaa actcttgcct aataccctgacagacaagtt ctatgtccat tctgagggaa 1500 cactagatat aaatggcgta actcccaaagaagggggttt atatacttgt atagcaacta 1560 acctagttgg cgctgacttg aagtctgttatgatcaaagt ggatggatct tttccacaag 1620 ataacaatgg ctctttgaat attaaaataagagatattca ggccaattca gttttggtgt 1680 cctggaaagc aagttctaaa attctcaaatctagtgttaa atggacagcc tttgtcaaga 1740 ctgaaaattc tcatgctgcg caaagtgctcgaataccatc tgatgtcaag gtatataatc 1800 ttactcatct gaatccatca actgagtataaaatttgtat tgatattccc accatctatc 1860 agaaaaacag aaaaaaatgt gtaaatgtcaccaccaaagg tttgcaccct gatcaaaaag 1920 agtatgaaaa gaataatacc acaacacttatggcctgtct tggaggcctt ctggggatta 1980 ttggtgtgat atgtcttatc agctgcctctctccagaaat gaactgtgat ggtggacaca 2040 gctatgtgag gaattactta cagaaaccaacctttgcatt aggtgagctt tatcctcctc 2100 tgataaatct ctgggaagca ggaaaagaaaaaagtacatc actgaaagta aaagcaactg 2160 ttataggttt accaacaaat atgtcctaaaaaccaccaag gaaacctact ccaaaaatga 2220 acaaaaaaaa aaaaagcgaa agactgcagttgtgctaaaa acaaaacaaa acaaacaaac 2280 aaaaaaaaaa 2290 5 316 PRT Homosapiens g1932712 5 Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr AsnPro Val 1 5 10 15 Ile Pro Phe Val Gly Thr Ile Pro Asp Gln Leu Asp ProGly Thr 20 25 30 Leu Ile Val Ile Arg Gly His Val Pro Ser Asp Ala Asp ArgPhe 35 40 45 Gln Val Asp Leu Gln Asn Gly Ser Ser Val Lys Pro Arg Ala Asp50 55 60 Val Ala Phe His Phe Asn Pro Arg Phe Lys Arg Ala Gly Cys Ile 6570 75 Val Cys Asn Thr Leu Ile Asn Glu Lys Trp Gly Arg Glu Glu Ile 80 8590 Thr Tyr Asp Thr Pro Phe Lys Arg Glu Lys Ser Phe Glu Ile Val 95 100105 Ile Met Val Leu Lys Asp Lys Phe Gln Val Ala Val Asn Gly Lys 110 115120 His Thr Leu Leu Tyr Gly His Arg Ile Gly Pro Glu Lys Ile Asp 125 130135 Thr Leu Gly Ile Tyr Gly Lys Val Asn Ile His Ser Ile Gly Phe 140 145150 Ser Phe Ser Ser Asp Leu Gln Ser Thr Gln Ala Ser Ser Leu Glu 155 160165 Leu Thr Glu Ile Val Arg Glu Asn Val Pro Lys Ser Gly Thr Pro 170 175180 Gln Leu Ser Leu Pro Phe Ala Ala Arg Leu Asn Thr Pro Met Gly 185 190195 Pro Gly Arg Thr Val Val Val Gln Gly Glu Val Asn Ala Asn Ala 200 205210 Lys Ser Phe Asn Val Asp Leu Leu Ala Gly Lys Ser Lys Asp Ile 215 220225 Ala Leu His Leu Asn Pro Arg Leu Asn Ile Lys Ala Phe Val Arg 230 235240 Asn Ser Phe Leu Gln Glu Ser Trp Gly Glu Glu Glu Arg Asn Ile 245 250255 Thr Ser Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu Met Ile Ile 260 265270 Tyr Cys Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His 275 280285 Ser Leu Glu Tyr Lys His Arg Phe Lys Glu Leu Ser Ser Ile Asp 290 295300 Thr Leu Glu Ile Asn Gly Asp Ile His Leu Leu Glu Val Arg Ser 305 310315 Trp

What is claimed is:
 1. An isolated and purified polynucleotide encodinga polypeptide comprising the amino acid sequence of SEQ ID NO:1.
 2. Anisolated and purified polynucleotide having a sequence which is fullycomplementary to the polynucleotide of claim
 1. 3. An isolated andpurified polynucleotide comprising the polynucleotide sequence of SEQ IDNO:3.
 4. An isolated and purified polynucleotide having a sequence whichis fully complementary to the polynucleotide of claim
 3. 5. Anexpression vector comprising the polynucleotide of claim
 1. 6. A hostcell comprising the expression vector of claim
 5. 7. A method forproducing a polypeptide, the method comprising the steps of: a)culturing the host cell of claim 6 under conditions suitable for theexpression of the polypeptide; and b) recovering the polypeptide fromthe host cell culture.
 8. A method for detecting a polynucleotide, themethod comprising the steps of: (a) hybridizing the polynucleotide ofclaim 2 to at least one nucleic acid in a sample, thereby forming ahybridization complex; and (b) detecting the hybridization complex,wherein the presence of the hybridization complex correlates with thepresence of the polynucleotide in the sample.
 9. The method of claim 8further comprising amplifying the polynucleotide prior to hybridization.